During the past years, two major advances in methodology have been made in our laboratory which will permit a more detailed and comprehensive investigation of neutrophil secretion. The first of these is the continued development and maturation of the permeabilized cell system as a model for secretion. We have been able to establish the intracellular Ca2+ requirements for secretion as well as the synergy seen between this divalent cation and guanine nucleotides. Preliminary data indicate that this system is well suited for the study of a variety of other aspects of neutrophil function, which are detailed in this application. In particular, we wish to look at the effects of osmolarity, solute composition, arachidonic metabolites, and multivalent cations on Ca2+-induced secretion. Furthermore, we wish to characterize the GTPase activity of permeabilized neutrophils and subcellular fractions as well as to investigate a novel GTP-utilizing "kinase" which we have recently discovered. Our second major technical advance has been the development of an in vitro model for studying the secretion proces itself. While much remains to be done in characterizing this simplified model system, we can eventually use it to study more unambiguously those factors which are required for membrane fusion and degranulation. In particular, we will complete the characterization of the plasma membrane fraction, such that we can optimize its orientation and/or integrity for fusion. In conjunction with this first goal, we will more fully characterize the Ca2+-ATPase of these membranes. Using the in vitro fusion system we will examine the roles of solutes and osmolarity as well as search for endogenous fusogens and synexins. Finally, we will be able to more directly examine the role of lipids and lipid metabolites in fusion. Our third specific aim is the development of monoclonal antibody probes. These will be used to both modulate the secretory process and to conduct cytochemical localization of components considered to be of interest in stimulus-secretion coupling. By comparing three different secretory systems (the intact neutrophil, the permeabilized cell, and in vitro fusion), each o which has a different level of integrity, structure, and complexity, it is hoped that major insights into the process of degranulation will be obtained with more certainty.